Significance: Epigenetic inactivation of tumor suppressor genes (TSGs) is a well-established event in cancer progression. TSG silencing is closely linked to aberrations in epigenetic mechanisms, including DNA methylation, histone modifications, and nucleosome positioning. The clinical success of DNA methylation inhibitors as a treatment for hematological malignancies has generated interest in expanding the use of these therapies. However, evidence suggests that epigenetic silencing is re-established when drug is withdrawn, often leading to disease reoccurrence and/or resistance to therapy. The broad objectives of this proposal are to determine how epigenetic alterations integrate in a gene-specific context to enforce aberrant epigenetic environments that silence or repress TSG expression. Specific Aims and Methods: Aim 1 is to determine the temporal sequence of molecular events associated with epigenetic resilencing. The primary goals of Aim 1 are to determine the temporospatial distribution of DNA methylation, histone modifications, and chromatin accessibility during the onset of TSG resilencing. Cultured cells will be treated with a DNA methyltransferase (DNMT) inhibitor to activate expression of epigenetically-silenced TSGs. Subsequent drug withdrawal potentiates TSG resilencing, which will be monitored in time course studies by novel epigenetic assays that we have developed. These studies will profile the appearance of epigenetic marks at four TSGs during the transition from active to repressed transcription versus two TSGs that remain active. A chief secondary objective is to develop an innovative epigenetic technology that enables investigators to assess directly if these changes map to the same or different molecules in a heterogeneous population of cells by linking chromatin immunoprecipitation and methyltransferase footprinting with bisulfite genomic sequencing. In Aim 2, Next Gen sequencing technologies will be optimized to simultaneously query DNA methylation and chromatin accessibility at 2,000 epigenetically-regulated promoters over a time course of gene resilencing. Coupled with gene expression microarray studies, this aim tests for global patterns of gene resilencing that may point to chromosomal position, gene ontology or mechanistic regulators. Aim 3 is to determine the extent to which epigenetic modifiers alter resilencing. The goals of this aim are to formulate working models of cause and effect in TSG silencing that will be tested by RNAi knockdown studies in pharmacologically de-repressed cells. These experiments are expected to discriminate between molecules responsible for maintenance or reprogramming of TSG silencing. Relevance: Elucidating epigenetic mechanisms that drive gene resilencing is crucial to a better understanding epigenetic dysfunction and therapy. Determining the temporal sequence of events leading to gene resilencing is expected to identify potential biomarkers of disease resistance. The mechanistic RNAi studies are expected to uncover new targets for therapeutic interventions and/or point to therapeutic strategies that will enhance the efficacy of clinically-used DNA methylation inhibitors.